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- Table of Contents
RAB27B / Ras-related protein Rab-27B · Western blot design guide
Design a Western Blot for RAB27B
Real validated RAB27B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAB27B WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
RAB27B Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~24.6 kDa | |
| Observed band | ~25 kDa | |
| Gel | 5–20% (catalog A04890-1) | |
| Positive control | Colon (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Acetylated + Methylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated RAB27B Western Blot Protocols
The A04890-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human MCF-7, human PC-3, human U-87MG (catalog A04890-1) |
| Gel % | 5–20% (catalog A04890-1) |
| Load | 30 ug; reducing conditions (catalog A04890-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A04890-1) |
| Membrane | nitrocellulose membrane (catalog A04890-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A04890-1) |
| Primary antibody | A04890-1 · 0.5 μg/mL (catalog A04890-1) |
| Primary incubation | overnight at 4°C (catalog A04890-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A04890-1) |
| Secondary incubation | 1.5 hour at RT (catalog A04890-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A04890-1) |
| Detection | ECL (catalog A04890-1) |
Section 2
What Is the Expected RAB27B Western Blot Band Size?
RAB27B is predicted at 24.6 kDa and observed at ~25 kDa; the cause of the small difference is not established.
| Single band at ~25 kDa | Matches the empirical RAB27B band and its 24.6 kDa predicted mass |
| ~25 kDa band in membrane-enriched material | Consistent with lipid-anchored RAB27B |
| Weak or absent band in a soluble fraction | RAB27B may remain associated with membranes |
| Mobility changes between reducing conditions | The listed intrachain disulfide may affect migration; the effect is unverified |
💡Expected RAB27B appearanceRAB27B has a predicted mass of 24.6 kDa and an empirical band at ~25 kDa; confirm band identity with ordinary controls.
| UniProt predicted mass | Places full-length RAB27B near 24.6 kDa; the empirical band is ~25 kDa |
| N-acetylthreonine at residue 2 | Modified terminus; no visible size effect is established |
| Cysteine methyl ester at residue 218 | Modified terminus; no visible size effect is established |
| Lipid anchoring and prenylation | May affect migration, but no size shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-associated RAB27B may be poorly extracted | Check membrane solubilization and a positive lysate control |
| Band higher than expected | Identity or migration difference is unestablished | Compare with the ~25 kDa positive control and verify antibody specificity |
| Band lower than expected | Identity or sample degradation is uncertain | Check sample integrity and compare with a positive control |
| Multiple bands | Additional band identities are unestablished | Compare with a positive control and use a RAB27B depletion control |
| Weak or no signal | Membrane-associated protein may be incompletely recovered | Check extraction and loading with a positive control |
Experiment evidence
Western blot images showing RAB27B band
A04890-1 · RAB27B
A04890-2 · RAB27B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RAB27B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RAB27B in Western blot, you can use colon tissue, which HPA rates as high expression.
Positive control: Colon (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Because RAB27B is membrane associated, membrane-enriched lysate may improve detection.
HPA tissue expression evidence for RAB27B
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Kidney | cells in tubules | High | Protein (IHC) | HPA → |
| Prostate | glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | glandular cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RAB27B Western Blot Tips
Deeper troubleshooting and optimisation questions for RAB27B, answered from its protein features.
How should RAB27B band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Could RAB27B isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It does not support assigning additional bands to RAB27B isoforms; verify their identity before interpreting them.
Which RAB27B modifications matter when interpreting bands?
PTM · UniProt lists N-acetylthreonine at position 2 and cysteine methyl ester at position 218. It also lists prenylation as a keyword without a site coordinate. These are UniProt coordinates; paper or antibody numbering may differ. Their presence alone does not establish a visible band shift.
Does this guide establish induction of RAB27B?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
What transfer method to use for RAB27B Western blot?
Transfer · RAB27B is approximately 25 kDa and membrane-associated. Use transfer conditions suitable for retaining a protein of that size, and verify transfer on the membrane. These features do not specify a uniquely optimal transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A04890-1 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should RAB27B be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why does RAB27B appear near 25 kDa?
Interpretation · Its predicted mass is 24.6 kDa, close to the observed ~25-kDa band. The listed modifications do not, by themselves, establish a visible shift or explain any difference between calculated and apparent mass.
RAB27B is listed as lipid-anchored at membranes and localized to late endosomes. Include the relevant membrane-containing material when preparing samples, and check whether your extraction retains membrane-associated RAB27B.
Because RAB27B is membrane-associated and found at late endosomes, compare like sample fractions and use consistent extraction and loading. A change in the measured band could reflect differences in recovery of membrane-associated protein.
The record lists one isoform, no signal or propeptide, and no glycosylation sites. It also lists two modified residues, one disulfide bond, and prenylation without a site coordinate. None of these features alone identifies an unexpected band; confirm band identity before assigning a cause.
RAB27B is listed as lipid-anchored at membranes and localized to late endosomes. Include the relevant membrane-containing material when preparing samples, and check whether your extraction retains membrane-associated RAB27B.
Because RAB27B is membrane-associated and found at late endosomes, compare like sample fractions and use consistent extraction and loading. A change in the measured band could reflect differences in recovery of membrane-associated protein.
The record lists one isoform, no signal or propeptide, and no glycosylation sites. It also lists two modified residues, one disulfide bond, and prenylation without a site coordinate. None of these features alone identifies an unexpected band; confirm band identity before assigning a cause.
Boster reagents
RAB27B Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-RAB27B Antibody Picoband®
Cat # A04890-1
Real WB data
Anti-RAB27B Antibody Picoband®
Cat # A04890-2
Both listed anti-RAB27B antibodies are rabbit polyclonals with Western blot images showing a band near the expected 25 kDa. A04890-1 was tested on selected human cell and rat and mouse stomach lysates; A04890-2 was tested on four human cell lysates. These examples do not establish universal sample performance.
Which to pick: Both list human, mouse, and rat reactivity. Choose A04890-1 for a blot example including all three species; choose A04890-2 if its human cell examples better match your sample. Both have WB images, but only A04890-1 shows rat and mouse samples.
Source: BosterBio RAB27B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O00194.
- Human Protein Atlas. RAB27B tissue expression.
- PMC10469664 — target-verified WB comparison
- PMC12093642 — target-verified WB comparison
- PMC3539959 — target-verified WB comparison